Mitochondrial Dynamics Imaging Constructs

AAV2/1.CMV.PI.Cre.RBG was purchased from Penn Vector Core (Philadelphia, PA). Drp1 shRNA-smCBA-mTagBFP2 was cloned into the pTR-CBA eGFP vector to replace eGFP [Drp1-targeting shRNA was a gift of S. Strack, University of Iowa (73 (link))]. The following constructs were previously cloned into the pCAGGS vector downstream of the chicken actin promoter: ATP-YEMK (gift of H. Noji, Osaka University) (74 (link)), Cre recombinase (2 (link)), EGFP-LC3 (Addgene), mitoGFP (4 (link)), and mitoTagBFP (2 (link)). mitoFarRed was generated by fusing TagRFP657 [gift from V. Verkhusha (Albert Einstein)] to the mitochondria-targeting sequence, cytochrome C oxidase subunit VIII (75 (link), 76 (link)), and mitoKeima was generated by cloning mito-mKeima from pIND-mito-mKeima (gift of A. Miyawaki) into pCAGGs. OM-GFP was made by fusing the OM-targeting sequence of rat Tom20 to GFP (77 (link)). GFP-Parkin was made by fusing GFP to Parkin (gift from A. Abeliovich, Columbia) and Matrix-roGFP from Addgene. pGW1-mitoEOS2 was cloned into the pGW1 from pEOS2-mito.

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Li H., Doric Z., Berthet A., Jorgens D.M., Nguyen M.K., Hsieh I., Margulis J., Fang R., Debnath J., Sesaki H., Finkbeiner S., Huang E, & Nakamura K. (2021). Longitudinal tracking of neuronal mitochondria delineates PINK1/Parkin-dependent mechanisms of mitochondrial recycling and degradation. Science Advances, 7(32), eabf6580.

Publication 2021

EGFP-LC3 mitoGFP

Actin Chicken Cre recombinase Cytochrome c oxidase Mito Mitochondria Parkin Shrna Subunit Vector

Corresponding Organization : University of California, San Francisco

Other organizations : University of California, Berkeley, Johns Hopkins Medicine, Johns Hopkins University

Top 5 similar protocols

Variable analysis

independent variables

Drp1 shRNA-smCBA-mTagBFP2
ATP-YEMK
Cre recombinase
EGFP-LC3
MitoGFP
MitoTagBFP
MitoFarRed
MitoKeima
OM-GFP
GFP-Parkin
Matrix-roGFP
PGW1-mitoEOS2

dependent variables

Not explicitly mentioned

control variables

Chicken actin promoter
PCAGGS vector
PTR-CBA eGFP vector

positive controls

Drp1-targeting shRNA (gift of S. Strack, University of Iowa)
ATP-YEMK (gift of H. Noji, Osaka University)
Cre recombinase
EGFP-LC3 (Addgene)
MitoGFP
MitoTagBFP
MitoFarRed (gift from V. Verkhusha, Albert Einstein)
MitoKeima (gift of A. Miyawaki)
OM-GFP
GFP-Parkin (gift from A. Abeliovich, Columbia)
Matrix-roGFP (Addgene)
PGW1-mitoEOS2

negative controls

Not explicitly mentioned

Annotations

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